One reason for the difficulty in developing effective treatments for myelodysplastic syndrome (MDS) is that there are no MDS cell lines which can be used to model or study the disease. Although numerous investigators have attempted to develop xenograft models for MDS, these attempts have been unable to engraft cells that will produce MDS in mice. Given that the NUP98-HOXD13 (NHD13) mice develop a highly penetrant MDS which closely resembles the human disease, we have begun studies to determine if these mice are a useful pre-clinical model to evaluate MDS therapies. Our initial studies have used the DNA-methyltransferase inhibitor 5-azacytidine; some of these results have recently been published (Genome Res. 4:580-91, 2014).However, since our initial studies used transgenic mice, effective treatment with 5-azacytidine could not replace the MDS bone marrow with completely normal (ie, wildtype or WT) bone marrow, since all of the bone marrow was transgenic. Therefore, in order to distinguish improvement in peripheral blood cytopenia due to differentiation of the MDS clone from elimination of the MDS clone, we have repeated the experiments using chimeric mice, that have both WT and NHD13 bone marrow. These repeat experiments have been performed using Decitabine (DAC), a related DNA methyltransferase inhibitor. Mice treated with DAC showed hematologic improvement and a survival advantage compared with saline-treated control mice; this experiment has now been repeated three times. Sorted BM cells from treated mice show clear differences in global cytosine methylation between the NHD13 and WT samples, and partial reversal of this hypermethylation with DAC treatment (collaboration with Dr. J.P. Issa). A manuscript describing these findings is in preparation. Drs. Difillipantonio, Doroshow, and colleagues from the Division of Cancer Treatment and Diagnosis (DCTD) have developed two novel DNMT1 inhibitors, one of which is now in phase I clinical trials. In collaboration with Dr. Difillipantonio and colleagues, we are now treating chimeric NHD13/WT mice with these compounds to assess efficacy in treatment of MDS. A manuscript describing the generation of chimeric mice with MDS was published in FY2019. Preliminary results show a survival benefit as well as improvement in hematopoiesis in mice receiving one of these DNMT1 inhibitors, designated T-dCyd. Portions of this data were presented at the 2018 ASH meeting, and a manuscript describing these findings is in preparation. The only curative option for patients with MDS remains allogeneic hematopoietic stem cell transplantation (HSCT). Allogeneic HSCT has two essential components, high-dose cytotoxic chemo-radiotherapy, and an immune-mediated graft versus host (GVH), or graft versus leukemia (GVL) effect. However, the relative contributions of high dose cytotoxic therapy and GVL are not well established in MDS patients. We propose to use transplantation of the NHD13 mice as a means to investigate this question. 1000 CGy induced a remission of 26-38 weeks, defined by normalization of peripheral blood counts and less than 2% circulating host cells. However, despite this period of prolonged remission, and prolonged survival compared to non-transplanted mice, all mice ultimately relapsed, indicating that this myeloablative therapy was not curative. To address the question of a GVL effect, we crossed C57bl6 mice with C3h.SW mice. C3H.SW mice are identical to C57Bl6 mice at the major histocompatibility loci, but have numerous mismatches at minor histocompatibility loci. For this reason, transplantation of C3H.SW donor cells into C57Bl6 host mice has been used to study GVH and GVL. We transplanted BM from C57Bl6/C3HSW mice into C57Bl6 NHD13 recipients. The mice developed little GVH or GVL under these conditions. Subsequent experiments using higher doses of BM or 5 x 10E06 peripheral T cells showed severe GVH. A third trial, using a higher dose of bone marrow cells (10E07)has led to survival 52 weeks post-transplant. These results demonstrate a survival benefit for bone marrow transplant; ongoing experiments are aimed at determining whether donor lymphocyte infusion or co-transplantation of specific T cell subsets will lead to yet greater survival. A portion of this data has been presented in abstract form and a manuscript describing these results was published in FY2018. In addition to the experiments outlined above, we have transferred NHD13 mice to colleagues at many academic institutions, and have licensed NHD13 mice to several biotech companies for pre-clinical studies. These colleagues have plans to treat NHD13 mice with a variety of agents, including histone deacetylase inhibitors, apoptosis inhibitors, and angiogenesis inhibitors. One of these compounds (ACE-536 or luspatercept) showed clinical activity in two phase III clinical trials for patients with MDS, and is currently being reviewed for approval by the FDA. As stated in the goals, we have generated leukemic mice and cell lines in which leukemia is driven by the binding of a leukemic fusion protein (NP23) to H3K4Me3 residues. We have treated these NP23 cell lines with compounds that have been shown to inhibit binding of PHD domains (including that present in the NP23 fusion) to H3K4Me3 in solution. NP23 cell lines are completely killed by a 12 hr treatment with one of these compounds (disulfiram), whereas control cell lines are not killed by this compound. Furthermore, cell death is associated with a marked (more than 5-fold) decrease in binding of the NP23 fusion protein to selected H3K4Me3 residues. In collaboration with Kura Oncology, we have begun testing novel inhibitors of the MLL-NUP98 axis, using NP23 and NUP98-NSD1 cell lines to evaluate the inhibitors.